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Plans for International Standard for HIV-2 RNA and 2 nd Second International Reference Panel for HIV-1 RNA Genotypes

Plans for International Standard for HIV-2 RNA and 2 nd Second International Reference Panel for HIV-1 RNA Genotypes. Harvey Holmes, Clare Morris and Neil Berry Division of Retrovirology NIBSC, UK. (In collaboration with Indira Hewlett, CBER/FDA).

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Plans for International Standard for HIV-2 RNA and 2 nd Second International Reference Panel for HIV-1 RNA Genotypes

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  1. Plans for International Standard for HIV-2 RNA and 2ndSecond International Reference Panel for HIV-1 RNA Genotypes Harvey Holmes, Clare Morris and Neil Berry Division of Retrovirology NIBSC, UK (In collaboration with Indira Hewlett, CBER/FDA)

  2. Current HIV Reference Reagents Available from NIBSC • 2nd International Standard for HIV-1 RNA. (code 97/650) • Unitage 5.56 IU Log 10/ml • International Reference Panel for HIV-1 RNA Genotypes (code 01/466) established in 2003 • No unitage assigned • Working reagents for NAT • Working Reagent 1 – medium copy number (3.56 log10 IU/ml) • Working Reagent 2 – high copy number (4.56 log10 IU/ml) • Working Reagent 3 – low copy number (2.56 log10 IU/ml) • Working Reagents and Proficiency Samples can also be made at NIBSC under contract to a pre-defined concentration and using various HIV-1 strains and genotypes

  3. International Standard for HIV-2 RNA • Rationale • Global HIV pandemic due to HIV-1 • Most NAT assays aimed at HIV-1 detection and quantification • HIV-2 mainly found in West Africa and European countries with close links – eg Portugal, France, Belgium. • Assays capable of detecting HIV-2 RNA or both HIV-1/HIV-2 are either available (eg Roche Cobas Taqscreen MPX) or in development • International Standard for the HIV-2 RNA component would be valuable once assays in wider use • Anticipated need/usage • Currently limited demand • Once commercial assays that can detect HIV-2 more available, demand may be greater • WHO Status • New project – endorsed by ECBS in October 2006 • Discussed at meeting of WHO Collaborating Centres in Jan 07

  4. Key Scientific issues • Few commercial assays available that can detect/quantitate HIV-2 RNA • At NIBSC, in house real-time PCR assay based on LTR sequence used for HIV-2 quantification • Source/Type of Material • To be based on HIV-2 grown in culture and diluted in negative plasma • Currently assessing representatives of HIV-2 subtype A and subtype B • Suitable strain(s) to be selected and stock laid down • Virus stock to be characterised and sequence confirmed • Heat inactivation to be evaluated • Batch to be freeze-dried • International collaborative study to be organised

  5. Amplification of HIV2 ROD log dilution series

  6. Standard Curve of HIV2 ROD standards

  7. 1st International Reference Panel for HIV-1 RNA Genotypes (code 01/466) • Established by ECBS in 2003 • 500 panels prepared and frozen at -80C • Contains representatives of HIV-1: • Subtypes A, B, C, D, AE, F, G, AGH • Group N and O • ~120 panels remain • Stability at -80C very good over 5 years • Proposal for 2nd IRP

  8. Second Genotype Panel for HIV-1 RNA • Rationale • Many inter-subtype recombinant forms now circulating (CRF01 – CRF32) • Increasingly being encountered – important pandemic strains • Less common subtypes may be difficult to detect • Proposal to prepare extended panel containing: • Subtypes G, H, J and K, group N and O • Range of CRFs • Will give kit manufacturers and others access to rare and challenging strains of HIV to enable them to assess their ability to detect these viruses • Anticipated need/usage • Discussed at SoGAT working group meeting • Limited need • Provides a source of well characterised diverse CRFs and isolates • WHO Status • Project endorsed by ECBS October 2006 • Discussed at meeting of WHO Collaborating Centres in Jan 07

  9. Second Genotype Panel for HIV-1 RNA • Source/Type of Material • Only relatively small number of characterised viruses belonging to uncommon subtypes or CRFs available as infectious virus • many only available as cloned DNA • Will source viruses from CFAR/NIBSC, CBER and NIH ARRRP and from scientists who have described them in the literature • Viruses to be grown in PBMC culture and seed stocks stored down • Viruses will be characterised including sequence confirmation • Viruses will be spiked into HIV-negative plasma • Heat inactivation and freeze-drying to be evaluated • International collaborative study to be organised

  10. Subtype G Subtype H Subtype J Subtype K Group N (to be sourced) Group O HIV-2 Subtype A HIV-2 subtype B CRF01_AE CRF02_AG CRF04_AGHKU (plasma only) CRF07_BC CRF11_A01GJ CRF13_A01GJU CRF14_BG HIV field isolates, reference strains and CRFs currently available

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